The invention relates to a device permitting the spreading of one or several reagents on a gel, in particular an electrophoresis or immunofixation gel.
The invention also relates to a method making use of the aforementioned device.
Electrophoresis is the migration of suspended or colloidal particles in a liquid or a gel, due to the effect of potential difference across immersed electrodes. Migration is toward electrodes of charge opposite that of the particles.
Electrophoresis is useful in the study of proteins because the protein molecules act like colloidal particles, their charge being positive or negative depending upon whether the surrounding solution is acidic or basic. Therefore, the acidity of the solution can be varied by the introduction of one or more reagents and used to control the direction in which a protein moves upon electrophoresis. Furthermore, different protein particles in a mixture move toward the electrodes with different velocities depending upon the number of charges carried by the particles. The techniques of electrophoresis or immunofixation are usually followed, after the migration, by a stage of incubating the gel with a reagent in order to detect and possibly to quantify the protein fractions separated during the electrophoresis.
The most conventional case is the staining of the set of protein fractions separated by electrophoresis by means of a dye which becomes bound specifically on to the latter. In this case, the reagent used (the dye) is a product which is generally cheap and of which it is possible to prepare a solution in a sufficient quantity to immerse the gel totally therein.
However, this method cannot be envisaged in the case where it is necessary to use small quantities of reagent, when it is an expensive reagent, or when it is desired to produce on the same gel incubations on discrete zones with different reagents.
These possibilities relate for example to assaying isoenzymes contained in a serum (isoLDH, isoCK, isophosphatase) where the gel is incubated with a substrate (expensive product) for these enzymes with the aim of forming a colored or fluorescent product which permits quantification by densitometry.
The case where it is necessary to carry out, on the same gel, incubations with different reagents is constituted for example by immunofixation where various discrete zones of the gel must be incubated with various antisera of different specificity (expensive reagent) and a protein fixative.
In all these cases, use of a minimum quantity of reagent is sought.
In implementing the abovementioned methods, a sheet of filter paper cut to the size of the gel is impregnated with a quantity of reagent just sufficient for it to be entirely moistened and it is then applied to the surface of the gel.
In the case where various reagents are possibly used on distinct zones of the same gel, as many strips of filter paper are cut as there are zones to be covered by the various reagents, and these various strips are impregnated with the various reagents before being applied to the various zones where the incubation is to take place.
This method has a certain number of drawbacks. It is in fact necessary to avoid trapping air bubbles between the gel and the sheet of filter paper impregnated with reagent, because, in the region of the air bubbles, the reaction would not take place for lack of reagent on the gel. The paper being opaque, it is sometimes difficult to detect the presence of these air bubbles.
Moreover, during the incubation reaction, a proportion of the proteins which it is desired to develop can be absorbed onto the paper, or more simply be absorbed by pumping onto the latter.
If an extended incubation time is necessary (30 minutes to 1 hour), this phenomenon is accentuated by the fact that, during the incubation, evaporation from the free surface of the paper occurs. The latter having a tendency, by capillarity, to maintain a constant degree of humidity, a flow of liquid is created, from the gel to the paper, with entrainment of the protein fractions to be assayed initially contained in the gel. This leads to a loss of sensitivity of the development on the gel and, this absorption not being very homogeneous, quantification by densitometry of the developed fractions risks being subject to errors.
Furthermore, and above all, this is a manual process which is very laborious and therefore difficult to implement in routine clinical analysis, and, in current practice, has been abandoned.
Another method also used consists in using a mask constituted by a sheet of flexible plastic, preferably hydrophobic in nature, having approximately the same external outline as the gel and having one or more independent openings of rectangular shape. This mask, once applied to the gel, delimits the zone or zones intended to receive the reactant or reactants.
Use of such a mask also has a certain number of drawbacks.
After electrophoretic migration, it frequently happens that a proportion of the liquid exudes from the surface of the gel. This exudation is mainly created by electroendosmotic flow.
Before applying the mask to the gel, it is necessary to remove this excess liquid by pumping by means of a thin filter paper, because, if liquid is still present on the gel, it prevents good adhesion of the mask to the gel, as well as a good seal at the periphery of the zone(s) delimited, being assured.
As well as the additional manual operation of pumping, the filter paper risks entraining a portion of the proteins from the gel and of distorting the result.
Furthermore, the gel or the reagents used can, in certain cases, contain surfactants which will facilitate insertion of liquid between the gel and the mask and, under certain conditions, even after removal of the excess liquid which has exuded from the gel at the end of migration, the mask risks not assuring the required seal.
With this type of mask, it is important that the whole of the surface of the mask is perfectly applied to the gel. It is necessary to avoid trapping air bubbles between the gel and the plastic mask during its positioning on the gel. Otherwise, there is a risk of having either a leak of reagent under the mask, or a mixing of adjacent reagents.
An object of the present invention is to propose a device which largely overcomes the preceding drawbacks.
The subject of the present invention is a device permitting deposition, spreading and incubation of a reagent on a gel, in which quantities of reagent of the same order of magnitude as those of conventional devices are used, the device of the invention being however without the drawbacks of the conventional devices.
Another object of the present invention is to propose a device permitting deposition, spreading and incubation of a reagent on a gel, in which it is possible to incubate different zones with respectively different reagents, without there being mixing of the reagents.
The aim of the present invention is to propose a device permitting deposition, spreading and incubation of a reagent on a gel, in which manual operations are limited to the minimum.
An object of the present invention is to propose a device for deposition, spreading and incubation of a reagent on a gel, permitting accurate results to be obtained as to quantification of the substances fractionated in the gel.
An object of the present invention is to propose a device for deposition, spreading and incubation of a reagent on a gel, capable of being automated.